Pharmacotherapy for vascular dysfuntion associated with deficient nitric oxide bioactivity

ABSTRACT

A patient with a disorder involving endothelial dysfunction associated with deficient nitric oxide bioactivity, e.g., coronary artery disease, atherosclerosis, hypertension, diabetes or neurodegenerative condition stemming from ischemia and/or inflammation, is treated by administering nitric oxide bioactivity increasing hydroxyguanidine.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/196,298, filed Apr. 12, 2000.

TECHNICAL FIELD

[0002] This invention is directed at enhancing vascular function inpatients with vascular diseases and conditions that are associated withdeficient nitric oxide bioactivity, endothelial dysfunction,tetrahydrobiopterin insufficiency and/or oxidative stress. In anembodiment the oxidative stress triggers the tetrahydrobiopterininsufficiency which in turn triggers deficient nitric oxide bioactivityand endothelial dysfunction, and the invention is directed at treatingthe vascular diseases and conditions associated with the endothelialdysfunction.

BACKGROUND OF THE INVENTION

[0003] It is known that nitric oxide is constitutively produced byvascular endothelial cells where it plays a key physiological role inthe moment-to-moment regulation of blood pressure and vascular tone.

[0004] It is known that deficient nitric oxide bioactivity contributesto the pathogenesis of vascular dysfunctions, including coronary arterydisease, atherosclerosis, hypertension, diabetic vasculapathy andneurodegenerative conditions stemming from ischemia and/or inflammation,and that such pathogenesis includes damaged endothelium, poor flow ofoxygenated blood resulting in oxygen-deficient organs and tissues,elevated systemic vascular resistance (high blood pressure), vascularsmooth muscle proliferation, progression of vascular stenosis andinflammation.

[0005] There is no current medically established solution for reversingor diminishing the deficiency in nitric oxide bioactivity. However,health food stores sell arginine and arginine-containing preparations asdietary supplements, and efficacy in reversing conditions associatedwith endothelial dysfunction has been suggested. Administration oftetrahydrobiopterin has also been suggested to increase nitric oxidebioactivity by blood vessels of chronic smokers and in animal models ofatherosclerosis.

SUMMARY OF THE INVENTION

[0006] It has been discovered in the course of making the invention thata predominant reason for nitric oxide (NO) deficiency in disordersinvolving endothelial dysfunction associated with deficient nitric oxidebioactivity is that dihydrobiopterin (BH₂) binds to eNOS (an enzymeassociated with constitutive nitric oxide production in endothelialcells of blood vessels) with affinity equal to the natural cofactortetrahydrobiopterin (BH₄), but that whereas BH₄-bound eNOS mediatesproduction of nitric oxide, BH₂-bound eNOS does not. Rather BH₂-boundeNOS causes diminished nitric oxide to be present by producingsuperoxide anion that reacts with nitric oxide to inactivate it.BH₂-bound eNOS also causes a cascade effect by producing superoxideanion which oxidizes BH₄ to BH₂ and still greater rate of superoxideproduction and further diminished production of nitric oxide andincreased inactivation of nitric oxide. Oxidative conditions that canpredominate in vascular disorders can oxidize BH₄ to BH₂, therebyinitiating this cascade. It is also discovered in the course of makingthe invention herein that hydroxyarginine, and otherhydroxyguanidine-containing molecules can be metabolized to nitric oxideby BH₂-bound eNOS.

[0007] The invention herein is directed to a method of treating apatient with a disorder involving endothelial dysfunction associatedwith deficient nitric oxide bioactivity by restoring or increasingnitric oxide bioactivity in the patient and comprises administering tothe patient a therapeutically effective amount of nitric oxidebioactivity increasing agent selected from the group consisting ofnitric oxide bioactivity increasing hydroxyguanidines andpharmaceutically acceptable salts thereof, optionally in combinationwith arginine and/or tetrahydrobiopterin, thereby increasing orrestoring nitric oxide bioactivity.

[0008] The endothelial dysfunction referred to is diagnosed by thefailure of intracoronary infusion of 1 μmol/liter of acetylcholine inphysiological saline to elicit an increase in coronary artery luminaldiameter in a patient undergoing coronary angiography. An alternativenon-invasive approach to assess endothelial dysfunction may be performedby measurement of flow-mediated vasodilation of the brachial arteryusing an ultrasound-based imaging technique. For this test, forearmbrachial artely diameter is determined by ultrasound in the patientprior to testing. Subsequently, a pneumatic tourniquet is placed belowthe patient's elbow, inflated to 300 mm Hg and held at this pressure for5 minutes. The tourniquet is then rapidly released and the flow-inducedincrease in luminal diameter is recorded at 1 min after release. If theobserved flow-induced increase in luminal diameter averages 5% or lesswith 4 repeat measurements, a diagnosis of endothelial dysfunction ismade.

[0009] The deficiency in nitric oxide bioactivity referred to above isdue to oxidative stress which oxidizes some of the normally presentnitric oxide and/or oxidizes tetrahydrobiopterin cofactor for nitricoxide production making it inactive, so as to deplete nitric oxidebioactivity.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010]FIG. 1 is a graph which compares the affinity that the pterins5,6,7,8-tetrahydrobiopterin (BH₄), 7,8-dihydrobiopterin (BH₂),6-methyltetrahydrobiopterin. (6MePH₄), and sepiapterin to compete for[³H] tetrahydrobiopterin binding to eNOS.

[0011]FIG. 2(a) shows electron paramagnetic resonance results thatassess superoxide production by BH₄-bound eNOS in buffer.

[0012]FIG. 2(b) shows electron paramagnetic resonance results thatassess superoxide production by sepiapterin-bound eNOS in assay buffer.

[0013]FIG. 2(c) shows electron paramagnetic resonance results ofsuperoxide production by BH₂-bound eNOS in assay buffer.

[0014]FIG. 3(a) is a graph showing BH₄-bound eNOS production of totalnitrate/nitrite from arginine in assay buffer withoutN^(G)-methyl-L-arginine (NMA), denoted “Control”; with NMA denoted“NMA”; and after elimination of calmodulin, denoted “No CaM.”

[0015]FIG. 3(b) is a graph showing BH₂-bound eNOS production of totalnitrate/nitrite from arginine in assay buffer without NMA, denoted“Control”; with NMA, denoted “NMA”; and after elimination of calmodulindenoted “No CaM.”

[0016]FIG. 3(c) is a graph showing BH₄-bound eNOS production of totalnitrate/nitrite from N^(ω)-hydroxyarginine in assay buffer without NMA,denoted “Control”; with NMA, denoted “NMA”, and after elimination ofcalmodulin, denoted “No CaM.”

[0017]FIG. 3(d) is a graph showing BH₂-bound eNOS production of totalnitrate/nitrite from N^(ω)-hydroxyarginine in assay buffer without NMA,denoted “Control”; with NMA, denoted “NMA”; and after elimination ofcalmodulin, denoted “No CaM.”

[0018]FIG. 4 is a graph containing curves showingconcentration-dependence of nitric oxide synthesis from N^(ω)-hydroxyarginine by BH₂-bound eNOS and by BH₄-bound eNOS.

[0019] The term “Specific Nitrite/Nitrate” in legends on FIGS. 3(a),3(b), 3(c), 3(d) and 4 means the increase of nitrite/nitrate observedabove background during the one hour incubation.

DETAILED DESCRIPTION

[0020] As indicated above, the method of the invention herein is fortreating a patient with a disorder involving endothelial dysfunctionassociated with deficient nitric oxide bioactivity by restoring orincreasing nitric oxide bioactivity in the patient and comprisesadministering to the patient a therapeutically effective amount ofnitric oxide bioactivity increasing agent selected from the groupconsisting of nitric oxide bioactivity increasing hydroxyguanidines andpharmaceutically acceptable salts thereof optionally in combination witharginine and/or tetrahydrobiopterin, thereby increasing or restoringnitric oxide bioactivity.

[0021] Disorders involving endothelial dysfunction associated withdeficient nitric oxide bioactivity are known and include coronary arterydisease, atherosclerosis, hypertension, diabetes and neurodegenerativeconditions stemming from ischemia and/or inflammation (e.g.,inflammatory and neurodegenerative conditions owing to insufficientnitric oxide production, e.g., stroke).

[0022] We turn now to the agents which are selected from the groupconsisting of nitric oxide bioactivity increasing hydroxyguanidines andpharmaceutically acceptable salts thereof.

[0023] The nitric oxide bioactivity increasing hydroxyguanidines arepreferably nitric oxide bioactivity increasing agents having theformula:

[0024] wherein R¹, R², R³ and R⁴ permit transport into cells and are thesame or different and can be independently selected from the groupconsisting of hydrogen, amino, imino, alkyl, substituted alkyl, phenyl,substituted phenyl cycloalkyl, benzyl, acyl, pyridyl, piperidyl,piperazyl, amino acid, lipid and carbohydrate and wherein R³ and R⁴ canoptionally join to form a ring. The alkyl can be, for example, C₁-C₁₀alkyl. The substituents on substituted alkyl include, for example, oneor more of the same or different of halo, thio, nitro, amino, carboxy,C₁-C₆ -alkoxy and aryl substituted on C₁-C₁₀ alkyl. The substituents onsubstituted phenyl include, for example, one or more of the same ordifferent of halogen, C₁-C₆ alkyl, nitro, amino and C₁-C₆ alkoxy (e.g.,methoxy). The cycloalkyl can contain, for example, from 3 to 8 carbonatoms. The acyl can be, for example, C₁-C₆ acyl. The halo and halogeninclude chloro, bromo and fluoro.

[0025] The pharmaceutically acceptable salts include, for example, thehydrochloride, acetate and sulfate salts. Other pharmaceuticallyacceptable salt group will be obvious to those skilled in the art.

[0026] Preferably at least one of R¹, R², R³ and R⁴ is hydrogen, andpreferably two or three of R¹, R², R³ and R⁴ are hydrogen.

[0027] When one or both of R³ and R⁴ are alpha-amino acids, thealpha-amino acid can be an L-compound or D-compound or D,L-compound.L-compounds are preferably used but D-compounds and D,L-compounds alsocan be used.

[0028] The hydroxyguanidine treating agents include, for example,N^(ω)-hydroxyarginine and hydroxyguanidine.

[0029] N^(ω)-Hydroxyarginine has the formula (I) where R¹, R² and R³ arehydrogen, and R⁴ is (CH₂)₃CH(NH₂)COOH.

[0030] Hydroxyguanidine has the formula (I) where R¹, R², R³ and R⁴ areall hydrogen.

[0031] N^(ω)-Hydroxyarginine and hydroxyguanidine are availablecommercially.

[0032] Still other hydroxyguanidine treating agents include, forexample, compounds of formula (I) where R¹, R² and R³ are H, and R⁴ is(CH₂)₃C(CH₃)(NH₂)COOH, e.g., N^(ω)-hydroxy-L-α-methylarginine; compoundsof the formula (I) were R¹, R² and R³ are H, and R⁴ is(CH₂)₄CH(NH₂)COOH, e.g., N^(ω)-hydroxy-L-homoarginine; compounds of theformula (I) where R¹, R² and R³ are H, and R⁴ is (CH₂)₄NH₂; andcompounds of the formula (I) where R¹, R² and R³ and H, and R⁴ is(CH₂)₄COOH.

[0033] The other hydroxyguanidines are prepared by methods well known inthe art from hydroxylamine or other simple precursors.

[0034] As indicated above, the agents are administered intherapeutically effective amounts, i.e., an endothelial dysfunctionreversing or diminishing effective amount that provides reversal ordiminishing or stopping of endothelial damage, increased oxygenatedblood flow to oxygen-deficient organs and tissues, diminished vascularresistance (increased blood vessel dilation), reversing or stopping ofprogression of vascular stenosis and/or diminished inflammation.Therapeutic amounts depend on the agent administered and can range, forexample, from 0.01 μmol/kg to 2 mmol/kg. For N^(ω)-hydroxyarginine,administration can be, for example, of a loading dose, e.g., of 20mg/kg, followed by 1 to 10 mg/kg/hr. Other suitable dosage informationfor N^(ω)-hydroxyarginine is exemplified in the working exampleshereinafter.

[0035] The routes of administration include oral, transdermal,intravenous, and intramuscular.

[0036] For transdermal administration, the agent can be administered,for example, as an ointment or cream containing from 0.1 to 3% of theagent.

[0037] Since the conditions treated are chronic, the administrationstypically are on a daily basis.

[0038] The mode of benefit includes improved flow of oxygenated blood tooxygen-deficient organs and tissue, reduced systemic vascularresistance, diminished progression of vascular stenosis, and diminishedinflammation.

[0039] We now turn to the optional case referred to above where agent asdescribed above is used in combination with administration of arginine.The arginine used is L-arginine. The L-arginine is used in atherapeutically effective amount which is an amount effective toincrease nitric oxide synthesis in vascular cells. This amount typicallyranges from 5 to 20 grams per day. The-L-arginine is preferablyadministered orally.

[0040] We turn now to optional case referred to above where agent asdescribed above is used in combination with administration oftetrahydrobiopterin. The tetrahydrobiopterin used is, for example,(6R)-5,6,7,8-tetrahydro-L-biopterin. The amount typically ranges from0.05 mg/kg to 10 mg/kg. The tetrahydrobiopterin is preferablyadministered orally.

[0041] The invention is supported by Reference Examples 1, 2, 3 and 4,and is illustrated by working Examples I, II, III, and IV and V whichare set forth below.

[0042] The eNOS used in Reference Examples 1, 2, 3 and 4 was made asdescribed in Martasek, P., et al., Biochem. Biophys. Res. Commn 219,359-365 (1996).

REFERENCE EXAMPLE 1

[0043] Increasing concentrations of unlabeled pterins were incubated for15 minutes at 22° C. with [³]tetrahydrobiopterin ([³H]BH₄), 10 pmoles,and eNOS, 3 pmoles, in binding buffer which is Tris.HCl, pH 7.5, (50mM), and dithiothreitol (DTT) (1 mM), in a 100 microliter volume in eachwell in a 96-well filtration plate assay. The pterins used include5,6,7,8-tetrahydrobiopterin (BH₄), 7,8-dihydrobiopterin (BH₂),6-methyltetrahydrobiopterin (6 MePH₄) and sepiapterin. Data which areshown in FIG. 1 are mean±SEM values of triplicate determinations.Similar results were obtained in four separate experiments. In FIG. 1,the squares denote BH₄ for the naturally occurring (R)-stereoisomer ofBH₄ and represent BH₄, the triangles represent BH₂, the diamondsrepresent 6 MePH₄, and the circles represent sepiapterin. In FIG. 1, theterm “Inhibitor” in the horizontal legend means pterin analog and isgeneric for BH₄, BH₂, 6MePH₄ and sepiapterin. Incubations were carriedout at the concentrations indicated by the data points in FIG. 1. Thisexperiment is to compare the ability of the named pterins to compete for[³H]BH₄ binding to eNOS. The results show that BH₂ and BH₄ bind withequal affinity to eNOS, so BH₂ formed in endothelial cells wouldeffectively compete for binding to eNOS with BH₄ and stop nitric oxideproduction in the cases where it binds to eNOS (as indicated inReference Example 3 and FIG. 3(b)).

REFERENCE EXAMPLE 2

[0044] Assay buffer utilized contained HEPES (50 mM, pH 7.4), calcium(0.2 mM), calmodulin (10 μg/ml), NADPH (0.1 mM), L-arginine (0.1 mM),tetrahydrobiopterin (10 μM), DEPMPO (structure shown in upper right ofFIG. 2(a)) (50 mM), and diethylenetriamine pentaacetic acid (DTPA) (0.1mM). Included was 7 pmol eNOS. Incubation was for 15 min at 22° C.Subsequent addition was either of sepiapterin (50 μM) or BH₂ (1 mM). TheDEPMPO functions as a probe (spin-trap) that selectively capturessuperoxide anion. Electron paramagnetic resonance (EPR) was carried outto determine superoxide production. EPR was carried out at microwavepower of 2 mW, modulation amplitude 1G, time constant 0.128 seconds,scan rate 1.6 G/s, gain 1.25×10E5, number of scans 10. EPR shows aneight peak signal when the DEPMPO captures superoxide. Results are shownin FIGS. 2(a), 2(b) and 2(c). The line under 20G in FIG. 2(c) indicatesthat horizontal distance represents 20 gauss in FIGS. 2(a), 2(b) and2(c). As shown in FIG. 2(a) eNOS does not produce superoxide when boundto BH₄. However, as shown in FIG. 2(c), subsequent addition of BH₂ candisplace BH₄ and activate superoxide production. The results withsepiapterin (FIG. 2(b)) support the conclusion that binding ofincompletely-reduced pterin, i.e., a dihydropterin such as sepiapterin,will activate superoxide production.

REFERENCE EXAMPLE 3

[0045] All samples were 100 microliter total volume and contained assaybuffer (Tris.HCl pH 7.6 (50 mM), DTT (1 mM), calcium (100 μM), andcalmodulin (100 nM)). In the assay buffer, eNOS (10 pmol), either BH₄(10 μM) or BH₂ (10 μM), and either L-arginine (100 μM) orN^(ω)-hydroxy-L-arginine (100 μM) were introduced. In some experiments,the nitric oxide synthase inhibitor N^(ω)-methyl-L-arginine (NMA, 1 mM)was additionally added or the required NO synthase cofactor calmodulinwas omitted (No CaM). Incubations were for 1 hour at 37° C. Totalnitrate/nitrite (as a measure of nitric oxide) was measured by theGreiss assay as described in “Methods of Nitric Oxide Research,” editedby Feelisch, M. and Stamler, J. S., John Wiley & Sons Ltd. (1996) atpages 491-497. Results are shown in FIGS. 3(a), 3(b), 3(c) and 3(d).FIG. 3(a) shows BH₄-bound eNOS production of total nitrate/nitrite fromarginine. FIG. 3(b) shows BH₂-bound eNOS production of totalnitrate/nitrite from arginine. FIG. 3(c) shows BH₄-bound eNOS productionof total nitrate/nitrite from N^(ω)-hydroxyarginine. FIG. 3(d) showsBH₂-bound eNOS production of total nitrate/nitrite fromN^(ω)-hydroxyarginine. Data represent means±SEM values of quadruplicatedeterminations. The results show that conversion to nitric oxide is by adifferent mechanism for arginine than for N^(ω)-hydroxyarginine in thatBH₂-bound eNOS does not cause production of nitric oxide from argininebut does cause production of nitric oxide from N^(ω)-hydroxyarginine,whereas BH₄-bound eNOS causes production of nitric oxide from botharginine and N^(ω)-hydroxyarginine. Although arginine conversion byBH₄-bound eNOS to nitric oxide is substantially blocked (>90%) byaddition of NMA or removal of CAM, N^(ω)-hydroxyarginine conversion tonitric oxide by BH₂-bound eNOS is little effected (<30%) by addition ofNMA or removal of CAM.

REFERENCE EXAMPLE 4

[0046] eNOS (10 pmol) was preincubated for 30 minutes at 37° C. in thepresence of either BH₂ (10 μM) or BH₄ (10 μM) in assay buffer (Tris.HClpH 7.6 (50 mM), DTT (1 mM), calcium (100 μM) and calmodulin (100 nM)).Then N^(ω)-hydroxy-L-arginine (denoted “Hydroxyarginine” in FIG. 4) wasadded (concentrations as disclosed in FIG. 4) and incubations were 100microliter total volume and were allowed to proceed for 1 hour at 37° C.Nitric oxide production was assessed from accumulation of its stableoxidation products (nitrite and nitrate), quantified by a modifiedGreiss assay (reference recited in Reference Example 3). The results areshown in FIG. 4. Data are mean±SEM values of quadruplicatedeterminations. The results show concentration dependence of nitricoxide synthesis from N^(ω)-hydroxyarginine by eNOS with either BH₂ orBH₄ as bound cofactor. Notably, N^(ω)-hydroxy-L-arginine supports theproduction of nitrogen oxides by eNOS in the presence of either BH₂ orBH₄.

EXAMPLE I

[0047] A 40-year-old male with Type I diabetes presents with symptoms ofpain in toes and loss of pink color (gray tissue tone) in toes. Anointment containing 1% by weight N^(ω)-hydroxy-L-arginine is applied tothe toes four times a day. Within 48 hours, pain diminishes and tissuebecomes pinker. Blood perfusion is increased.

EXAMPLE II

[0048] A 60-year old male with coronary artery disease develops chestpain and electrocardiographic evidence of angina after 10 minutes on atreadmill at 3 mph and 5% incline. Within 90 minutes after an oral doseof 10 mg/kg of N^(ω)-hydroxy-L-arginine, the subject is able to walk onthe treadmill at 3 mph and 5% incline for 25 minutes without pain orevidence of arginine.

EXAMPLE III

[0049] A 60-year-old female has moderate hypertension (150/100 mm Hg)and elevated vascular resistance. One hour after receiving a single 10mg/kg intravenous dose of N^(ω)-hydroxy-L-arginine, mean arterial bloodpressure is diminished by 14 mm Hg and systemic vascular resistance isreduced by 10%.

[0050] In the above Examples I, II, and III, a therapeutically effectiveamount of other hydroxyguanidine-containing agents can be substitutedfor the N^(ω)-hydroxy-L-arginine to obtain the benefits of improvedoxygenated blood flow to oxygen-deficient organs, lessened symptoms ofcoronary artery disease, reduced systemic vascular resistance,diminished progression of vascular stenosis and diminished inflammation.

EXAMPLE IV

[0051] A 55-year-old man suffers from type 2 diabetes (adult onsetdiabetes or insulin resistant diabetes), coronary artery disease andhypertension (a not uncommon composite of conditions), experiences astroke resulting in acute left-sided paralysis owing to right middlecerebral artery occlusion. Surviving the stroke, the patient is placedon chronic oral therapy with N^(ω)-hydroxy-L-arginine, 5 mg/kg every 4hours, or a combination of this with L-arginine, 20 mg/kg every 4 hours.There is no recurrence of stroke within the next two years.

EXAMPLE V

[0052] A 60-year-old man exhibits mild hypertension (140/90 mm Hg) andangiographic evidence of coronary artery atherosclerosis and familialhistory of cardiovascular disease. He is treated with 5 mg/kgN^(ω)-hydroxy-L-arginine every 4 hours orally as either free drug or inadmixture with antioxidant agents and vitamins (e.g., ascorbate,alpha-tocopherol, vitamin B6, vitamin B12, folate (folic acid),carotenoids, coenzyme Q10, phytoestrogens (including isoflavonoids),selenium, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA)and n-3 polyunsaturated fatty acids (PUFA)) with or without L-argininesupplementation (20 mg/kg every 4 hours). The mixture is delivered as anutriceutical. Blood pressure normalizes to less than 130/80 mm Hg andangiographic evidence indicates atherosclerosis progression is less than10% additional over the next five years.

[0053] When (6R)-5,6,7,8-tetrahydrobiopterin, 300 mg is substituted forthe L-arginine in Examples IV and V, results of no recurrence of strokewithin the next two years and normalized blood pressure, are obtained.

[0054] Variations

[0055] Variations in the above will be evident to those skilled in theart. Thus, the scope of the invention is defined by the claims.

What is claimed is:
 1. A method for treating a patient with a disorderinvolving endothelial dysfunction associated with deficient nitric oxidebioactivity, comprising administering to the patient a therapeuticallyeffective amount of agent which is selected from the group consisting ofnitric oxide bioactivity increasing hydroxyguanidines andpharmaceutically acceptable salts thereof.
 2. The method of claim 1where said agent is a nitric oxide bioactivity increasing compoundhaving the formula:

wherein R¹, R², R³ and R⁴ permit transport in cells and are the same ordifferent and can be independently selected from the group consisting ofhydrogen, amino, imino, alkyl, substituted alkyl, phenyl, substitutedphenyl, cycloalkyl, benzyl, acyl, pyridyl, piperidyl, piperazyl, aminoacid, lipid or carbohydrate and where R³ and R⁴ can optionally join toform a ring.
 3. The method of claim 2 where the disorder is selectedfrom the group consisting of coronary artery disease, atherosclerosis,hypertension and diabetes.
 4. The method of claim 3 where the agent isN¹⁰⁷ -hydroxy-L-arginine.
 5. The method of claim 1 where atherapeutically effective amount of L-arginine is also administered. 6.The method of claim 1 where a therapeutically effective amount oftetrahydrobiopterin is also administered.